Site-targeted microbubbles (MB) have demonstrated potential as a molecular imaging tool. A new targeted MB platform was developed in phase I of this project. This MB enables covalent coupling of targeting ligands using thioether conjugation chemistry. MB bearing a novel single-chain VEGF (scVEGF) targeting ligand was synthesized. The scVEGF MB bind with high specificity to endothelial VEGF receptors, and do not activate VEGFR signaling. These agents have application as a molecular imaging probe in a research setting, and have potential for translation to clinical use. We now seek to validate this agent as a research tool for molecular ultrasound imaging in drug discovery and basic science, and to investigate a potential clinical use. We hypothesize that scVEGF MB can detect angiogenesis in the context of tumor growth with greater sensitivity and specificity than non-targeted MB, B-mode ultrasound alone, or optical imaging probes. Specifically, we will evaluate the relationship between targeted microbubble concentration and quantified acoustic signal in vitro and in vivo, examine the ability of scVEGF MB to detect angiogenic tumors in several mouse models of cancer, and investigate the ability of these agents monitor response to therapy. Additionally, we will explore the potential utility of scVEGF MB in the context of prostate cancer biopsy guidance. The relationship between acoustic signal and targeted microbubble concentration will first be analyzed in vitro with an ultrasound flow phantom using a novel real-time ultrasound contrast quantification scheme. The sensitivity and specificity of scVEGF MB for angiogenic tumor vasculature will be examined in a bioluminescent mouse model of cancer, and validated by immunohistochemistry and bioluminescence imaging. The utility of scVEGF MB as a tool for monitoring response to therapy will be examined in a mouse model of prostate cancer. The change in tumor volume over several weeks of treatment will be evaluated using calipers, and correlated to the quantified scVEGF MB signal. Additionally, the sensitivity of scVEGF MB for monitoring treatment response will be compared against non-targeted MB (which quantify tumor blood flow) and a near-infrared VEGFR2 optical imaging contrast agent. Prostate biopsy guidance is a potential clinical application for the scVEGF MB, and we will evaluate feasibility for this in a canine model of the disease. Prostate biopsy of canines referred to a veterinary imaging center will be performed under standard B-mode ultrasound or scVEGF MB guidance, and the ability of scVEGF MB guidance to enhance prostate cancer detection will be examined. We anticipate that this project will enable determination of whether scVEGF MB can 1) specifically image angiogenic tumors, 2) monitor the response to experimental anti-cancer therapies in relevant preclinical models, and 3) increase the sensitivity of biopsy guidance in a clinical setting. These studies will enable validation of scVEGF microbubbles as a product for detecting and monitoring tumor development in a preclinical research and drug discovery setting, and evaluate the potential for this technology in a clinical setting. PUBLIC HEALTH RELEVANCE: Probes able to detect molecular components of cancer have the potential to accelerate drug discovery, enhance basic research, and improve the sensitivity of cancer detection in a clinical setting. Ultrasound imaging is widely used in both research and clinical applications, and provides a cost-effective, real-time imaging technique that does not require ionizing radiation. The tumor-targeted agent proposed here will enable the use of ultrasound imaging for molecular imaging of cancer with greater sensitivity and safety than existing technology.